Since September 2000, when Dr. Markovitz joined CBER's Division of Cellular and Gene Therapies, Dr. Markovitz's lab has investigated disease development due to infections by herpes simplex viruses (HSV). Samples of HSV isolated from patients can infect and replicate in a broad range of cell types and are thus associated with a wide range of diseases in the human population. Several strains of herpes simplex virus that have been modified with the intention of inhibiting replication (attenuated strains) are currently being investigated for use as tumor vaccines and in gene therapy of neurological diseases. Previously Dr. Markovitz and others have shown that one of these strains can replicate only weakly in many normal brain cell types but still can replicate in other cell types (e.g. tumor cells, ependymal cells). The goal of Dr. Markovitz's research is to identify 1) the kinds of normal cell types in which other attenuated HSV strains replicate, and 2) the cellular and viral factors involved in the replication of these strains in the identified types of cells. The knowledge gained from these studies will contribute to a science-based evaluation of the risks involved in using HSV vectors in gene therapy. Herpes simplex virus-1 (HSV-1) infects and replicates in a broad range of cell types and is thus associated with a wide range of disease. Within the nervous system, HSV-1 commonly establishes a latent infection in peripheral neurons. This active infection is suppressed by an immunocompetent host immune response. However, HSV infection of the central nervous system (CNS) can cause herpes simplex encephalitis that, especially in immune-impaired individuals, results in death or severe and long-lasting neurological sequelae. Replication-competent neuroattenuated HSV-1 vectors-- currently used in clinical trials for the treatment of cancers including brain tumors -- are reported to replicate poorly in the CNS. This research program focuses on 1) the ability of these and other HSV vectors to replicate productively in normal cell types in vivo, 2) the cellular and viral factors important for robust productive replication of these vectors, and 3) the effect of infection and associated immune response on neuronal function. This program has identified at least one normal brain cell type in the mouse that supports, in vivo, the productive replication of HSV vectors deleted in the gene encoding ICP34.5. The reason why these viruses replicate in this normal cell type is being explored. Studies focusing on cellular factors involved in viral replication and pathogenesis have progressed as well. During late stages of cellular infection by HSV, a nuclear structure is formed that contains at least 4 viral proteins: UL3, UL4, UL20.5 and ICP22/Us1.5. Of these four proteins, the function of only ICP22/Us1.5 is in part understood. Importantly, ICP22 is thought to be involved in neurovirulence; the lethal dose (LD50) as measured by intracranial inoculation of mice with viruses deleted in ICP22 is much higher than that observed with wild type virus. In the last few years we have identified the transcript responsible for translation of UL3, constructed novel viruses functionally deleted for the UL3 protein and are finishing construction of a virus for evaluation of the neurovirulence of UL3 in in vivo models. We have identified one cellular protein that interacts with the form of UL3 expressed in virally infected cells and future work will identify additional cellular protein that are contained in the aforementioned nuclear structures.